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PubMed1997

[Mass development of Stachybotrys chartarum on compostable plant pots made from recycled paper].

After handling plants grown in decomposable pots made of recycling paper, three women working in a big horticulture developed very painful inflammated efflorescences at the finger-tips, followed by scaling off the skin. The pots appeared to be very mouldy. Black masses of conidia of Stachybotrys chartarum and perithecia of Chaetomium globosum were identified on almost every pot. Apart from various other fungal genera, Trichoderma und Acremonium were frequently detected. Considering the observed symptoms, special attention was payed to the mycotoxin producing species St. chartarum. To evaluate the inhalative spore load, air sampling was performed. The detection of St. chartarum in the air was only possible with the spore trap (sampling of particles) but not with the Andersen sampler (detection of colony forming units). Without moving the pots, measurements yielded values of 30-100 St. chartarum conidia per m3 of air. The concentration of air-borne conidia increased drastically by handling the pots, thus attaining up to 7,500 conidia per m3 of air for St. chartarum only. The occurrence of St. chartarum in such amounts is alarming because of possible toxin production. In addition, the allergenic stress by fungal spores has to be emphasized. The results are discussed with regard to general medical-mycological aspects related to the degradation of environmentally-friendly decomposable materials.

Acremonium↗

PubMedDate not supplied

[Joint immobilization of L-lysine-alpha-oxidase and peroxidase on porous membrane carriers].

The goal of the present study was the development of the optimal method of co-immobilization of two enzymes: L-lysine alpha-oxidase from Trichoderma sp. and horseradish peroxidase. Commercial nitrocellulose, nylon and N+ nylon membranes were used as carriers. The immobilization was carried out either by absorbtion or by covalent binding with aldehyde groups. The aldehyde groups were attached to the surface of the carriers by UV-irradiation of membranes in the presence of p-azidotetrafluorobenzaldehyde. The optimal concentrations of reagents, enzymes and reaction conditions were found. The membranes with the co-immobilised L-lysine alpha-oxidase and peroxidase were shown to be useful for the determination of L-lysine concentrations.

Amino Acid Oxidoreductases↗

PubMed1976

Organic amendments and control of foot rot of Piper betle caused by Phytophthora parasitica var. piperina.

Experiments have been performed to control foot rot of Piper betle by organic amendments and antagonists in soil. Soil amended with corn straw gave best control. Corn straw supplemented with nitrogen (NH4NO3) gave still better control. The effect of different antagonists growing on corn-straw and til (Sesamum indicum) oil cake amendments showed that Trichoderma viride gave best control. Aspergillus terreus failed to control the disease although it showed great antifungal activity under controlled condition in Petri dishes.

Antibiosis↗

PubMed1998

Hydrolysis of cellulose using ternary mixtures of purified cellulases.

The saccharification of microcrystalline cellulose by reconstituted ternary mixtures of purified cellulases (one endoglucanase and two cellobiohydrolases) has been studied over the entire range of mixture compositions. Ternary plots are used to compare the performance of five synthetic mixtures drawn from the cellulase systems of Acidothermus cellulolyticus, Trichoderma reesei, Thermomonospora fusca, and Thermotoga neapolitana. Results reveal that at least one synthetic mixture utilizing enzymes from three different organisms delivers performance competitive with that of a "native" (i.e., co-evolved) ternary system drawn exclusively from T. reesei. This heterologous system, consisting of the endoglucanase E1 from A. cellulolyticus and the exoglucanases CBHI from T. reesei and E3 from T. fusca, is forgiving from the system-design point of view, in that it delivers high saccharification rates over a wide range of mixture compositions.

Aspergillus niger↗

PubMed1999

Crystal structure of the catalytic core domain of the family 6 cellobiohydrolase II, Cel6A, from Humicola insolens, at 1.92 A resolution.

The three-dimensional structure of the catalytic core of the family 6 cellobiohydrolase II, Cel6A (CBH II), from Humicola insolens has been determined by X-ray crystallography at a resolution of 1.92 A. The structure was solved by molecular replacement using the homologous Trichoderma reesei CBH II as a search model. The H. insolens enzyme displays a high degree of structural similarity with its T. reesei equivalent. The structure features both O- (alpha-linked mannose) and N-linked glycosylation and a hexa-co-ordinate Mg2+ ion. The active-site residues are located within the enclosed tunnel that is typical for cellobiohydrolase enzymes and which may permit a processive hydrolysis of the cellulose substrate. The close structural similarity between the two enzymes implies that kinetics and chain-end specificity experiments performed on the H. insolens enzyme are likely to be applicable to the homologous T. reesei enzyme. These cast doubt on the description of cellobiohydrolases as exo-enzymes since they demonstrated that Cel6A (CBH II) shows no requirement for non-reducing chain-ends, as had been presumed. There is no crystallographic evidence in the present structure to support a mechanism involving loop opening, yet preliminary modelling experiments suggest that the active-site tunnel of Cel6A (CBH II) is too narrow to permit entry of a fluorescenyl-derivatized substrate, known to be a viable substrate for this enzyme.

Catalytic Domain↗

PubMed1998

Three-dimensional structure of the ion-channel forming peptide trichorzianin TA VII bound to sodium dodecyl sulfate micelles.

Trichorzianin TA VII, Ac0 U1 A2 A3 U4 J5 Q6 U7 U8 U9 S10 L11 U12 P13 V14 U15 I16 Q17 Q18 Fol19, is a nonadecapeptide member of the peptaibol antibiotics biosynthesized by Trichoderma soil fungi, which is characterized by a high proportion of the alpha, alpha-dialkylated amino acids, alpha-aminoisobutyric acid (Aib, U) and isovaline (Iva, J), an acetylated N-terminus and a C-terminal phenylalaninol (Pheol, Fol). The main interest in such peptides stems from their ability to interact with phospholipid bilayers and form voltage-dependent transmembrane channels in planar lipid bilayers. In order to provide insights into the lipid-peptide interaction promoting the voltage gating, the conformational study of TA VII in the presence of perdeuterated sodium dodecyl sulfate (SDS-d25) micelles has been carried out. 1H sequential assignment have been performed with the use of two-dimensional homo- and -heteronuclear nmr techniques including double quantum filtered correlated spectroscopy, homonuclear Hartmann-Hahn, nuclear Overhauser effect spectroscopy, 1H-13C heteronuclear single quantum correlation, and heteronuclear multiple bond correlation. Conformational parameters, such as 3JNHC alpha H coupling constants, temperature coefficients of amide protons (delta gamma/delta TNH) and quantitative nuclear Overhauser enhancement data, lead to detailed structural information. Ninety-eight three-dimensional structures consistent with the nmr data were generated from 231 interproton distances six phi dihedral angle restraints, using restrained molecular dynamics and energy minimization calculations. The average rms deviation between the 98 refined structures and the energy-minimized average structure is 0.59 A for the backbone atoms. The structure of trichorzianin TA VII associated with SDS micelles, as determined by these methods, is characterized by two right-handed helical segments involving residues 1-8 and 11-19, linked by a beta-turn that leads to an angle about 90 degrees-100 degrees between the two helix axes; residues 18 and 19 at the end of the C-terminal helix exhibit multiple conformations.

Amino Acid Sequence↗

PubMed1998

Factorial optimization of a six-cellulase mixture

A factorial experimental design approach was used to optimize mixtures of six cellulases (five Thermomonospora fusca cellulases and plus/minus Trichoderma reesei CBHI along with beta-glucosidase) so as to maximize the glucose produced from filter paper. Optimized mixture A and mixture B produced glucose at 25 and 8.3 μmol glucose/μmol enzyme/min, respectively, which are 8 and 1.5 times higher than the sum of the activity of the individual cellulases. In both mixtures, the glucose yield depended on the ratio and the cellulases used. Most enzymes showed synergistic interactions that increased the glucose yield. The yield of glucose with the optimum mixtures depended on the total enzyme concentration. Copyright 1998 John Wiley & Sons, Inc.

Journal Article↗

PubMed1998

Improved immobilization of fusion proteins via cellulose-binding domains.

Cellulose-binding domains (CBDs) are structurally and functionally independent, noncatalytic modules found in many cellulose or hemicellulose degrading enzymes. Recent biotechnological applications of the CBDs include facilitated protein immobilization on cellulose supports. In some occasions there have been concerns about the stability of the CBD driven immobilization. Here we have studied the chromatographic behavior of variants of the Trichoderma reesei cellobiohydrolase I CBD belonging to family I. Both CBDs fused to antibody fragments and isolated CBDs were studied and compared. Tritium labeling by reductive methylation was used as a sensitive detection method. The fusion protein as well as the isolated CBD was found to leak from the column at a rate of 0.3-0.5% of the immobilized protein per column volume. However, the leakage could be overcome by using two CBDs instead of a single CBD for the immobilization. In this way leakage was reduced to less than 0.01% per column volume. The improved immobilization could also be seen as a decreased migration of the protein down the column in extended washes.

Binding Sites↗

PubMed1999

Total synthesis and membrane modifying properties of the lipopeptaibol trikoningin KB II and its analogues with acyl chains of different length at the N- and C-termini.

Trikoningin KB II, a ten-amino acid residue lipopeptaibol blocked at the N-terminus by the n-octanoyl group and at the C-terminus by the 1,2-amino alcohol L-leucinol, and extracted from the fungus Trichoderma koningii, exhibits membrane-modifying properties. We have synthesized by solution-phase methods trikoningin KB II and several analogues with acyl chains of different length at the N- and C-termini. Permeability measurements showed that an appropriate length of the linear acyl chain is a more important characteristic for the onset of significant membrane-modifying activity than its position in the peptide chain.

Amino Acid Sequence↗

PubMed2001

Synthesis and antifungal activity of 3,3'-ethylenebis(5-alkyl-1,3,5-thiadiazine-2-thiones).

In a search for promising antifungal compounds, nine new 3,3'-ethylenebis(5-alkyl-1,3,5-thiadiazine-2-thiones) were synthesized by the reaction of ethylene diamine, carbon disulfide, formaldehyde, and the appropriate alkyl amine. The title compounds were tested for their antifungal activity in vitro against pathogenic (Trichophyton rubrum and Candida albicans), phytopathogenic (Penicillum expansum, Trichoderma hazianum, and Fasarium oxysporum), and aflatoxin producing (Aspergillus flavus) fungi. These compounds exhibited varied inhibitory effects on growth or sporulation of some tested fungal species.

Antifungal Agents↗

PubMed2002

New 2H-tetrahydro-1, 3, 5-thiadiazine-2-thiones incorporating glycine and glycinamide as potential antifungal agents.

The new title derivatives (4b-h and 5a-i) were synthesized by reaction of the appropriate primary amine, carbon disulphide, and formaldehyde. These derivatives were prepared in order to study the effects of introducing polar groups at N3 or N5 or at both positions on the biological activity. The compounds were tested for their antifungal activity in vitro against pathogenic (Trichophyton rubrum and Candida albicans), phytopathogenic (Penicillum expansum, Trichoderma hazianum, and Fasarium oxysporum), and aflatoxin-producing (Aspergillus flavus) fungi. These compounds exhibited varied inhibitory effects on growth or sporulation of some tested fungal species.

Antifungal Agents↗

PubMed2001

The metabolic burden of the PGK1 and ADH2 promoter systems for heterologous xylanase production by Saccharomyces cerevisiae in defined medium.

Five recombinant S. cerevisiae strains were cultivated under identical conditions to quantify the molecular basis of the metabolic burden of heterologous gene expression, and to evaluate mechanisms for the metabolic burden. Two recombinant S. cerevisiae strains, producing Trichoderma reesei xylanase II under control of either the PGK1 or ADH2 promoters, were compared quantitatively with three references strains, where either the heterologous xylanase II (XYN2) gene, or the heterologous gene and the promoter and terminator were omitted from the recombinant plasmid. Neither the replication of multiple copies of the 2-microm-based YEp352 plasmid nor the replication the foreign XYN2 gene represented a metabolic burden to the cell, as the growth of the host organism was not affected. The inclusion of a glycolytic promoter on the recombinant plasmid, however, reduced the maximum specific growth rate (12% to 15%), biomass yield on glucose (8% to 11%), and specific glucose consumption rate (6% to 10%) of the recombinant strains. The presence of the heterologous XYN2 gene on the recombinant plasmid caused a further reduction in the maximum specific growth rate (11% to 14%), biomass yield (4%), and specific glucose consumption rate (12%) of the host strain during active gene expression, which was dictated by the regulatory characteristics of the promoter utilized. The metabolic effect of foreign gene expression was disproportionally large, with respect to on the amount of heterologous protein produced. This was most likely due to an increased energetic demand for the expression of a foreign gene and/or a competition for limiting amounts of transcription or translation factors, biosynthetic precursors or metabolic energy.

Alcohol Dehydrogenase↗

PubMed2004

Microplate-based filter paper assay to measure total cellulase activity.

The standard filter paper assay (FPA) published by the International Union of Pure and Applied Chemistry (IUPAC) is widely used to determine total cellulase activity. However, the IUPAC method is not suitable for the parallel analyses of large sample numbers. We describe here a microplate-based method for assaying large sample numbers. To achieve this, we reduced the enzymatic reaction volume to 60 microl from the 1.5 ml used in the IUPAC method. The modified 60-microl format FPA can be carried out in 96-well assay plates. Statistical analyses showed that the cellulase activities of commercial cellulases from Trichoderma reesei and Aspergillus species determined with our 60-microl format FPA were not significantly different from the activities measured with the standard FPA. Our results also indicate that the 60-microl format FPA is quantitative and highly reproducible. Moreover, the addition of excess beta-glucosidase increased the sensitivity of the assay by up to 60%.

Cellulase↗

PubMed2007

Application of cellulases from an alkalothermophilic Thermomonospora sp. in biopolishing of denims.

Use of cellulase for denim washing is a standard eco-friendly technique to achieve desirable appearance and softness for cotton fabrics and denims. But enzymatic washing of denim till date involved acid cellulase (Trichoderma reesei) and neutral cellulase (Humicola isolens) the use of which has a drawback of backstaining of the indigo dye on to the fabric. Though it has been suggested that pH is a major factor in controlling backstaining there are no reports on use of cellulase under alkaline conditions for denim washing. In this study for the first time an alkali stable endoglucanase from alkalothermophilic Thermomonospora sp. (T-EG) has been used for denim biofinishing under alkaline conditions. T-EG is effective in removing hairiness with negligible weight loss and imparting softness to the fabric. Higher abrasive activity with lower backstaining was a preferred property for denim biofinishing exhibited by T-EG. The activities were comparable to acid and neutral cellulases that are being regularly used. The enzyme was also effective under non-buffering conditions which is an added advantage for use in textile industry. A probable mechanism of enzymatic finishing of cotton fabric has been represented based on the unique properties of T-EG.

Actinomycetales↗

PubMed2007

Design of highly efficient cellulase mixtures for enzymatic hydrolysis of cellulose.

An extremely highly active cellobiohydrolase (CBH IIb or Cel6B) was isolated from Chrysosporium lucknowense UV18-25 culture filtrate. The CBH IIb demonstrated the highest ability for a deep degradation of crystalline cellulose amongst a few cellobiohydrolases tested, including C. lucknowense CBH Ia, Ib, IIa, and Trichoderma reesei CBH I and II. Using purified C. lucknowense enzymes (CBH Ia, Ib, and IIb; endoglucanases II and V; beta-glucosidase, xylanase II), artificial multienzyme mixtures were reconstituted, displaying an extremely high performance in a conversion of different cellulosic substrates (Avicel, cotton, pretreated Douglas fir wood) to glucose. These mixtures were much or notably more effective in hydrolysis of the cellulosic substrates than the crude multienzyme C. lucknowense preparation and other crude cellulase samples produced by T. reesei and Penicillium verruculosum. Highly active cellulases are a key factor in bioconversion of plant lignocellulosic biomass to ethanol as an alternative to fossil fuels.

Cellulase↗

PubMed2005

Analysis of the lipophilic peptaibol alamethicin by nonaqueous capillary electrophoresis-electrospray ionization-mass spectrometry.

The microheterogeneous peptaibol alamethicin F30 isolated from the culture broth of Trichoderma viride was analyzed by nonaqueous CE-electrospray-MS using an IT and a TOF mass analyzer. Compared to aqueous buffers, higher separation selectivity was observed for methanolic BGE allowing the detection of more minor components. The low electrophoretic mobility observed for neutral analytes under nonaqueous conditions may be explained by ion-dipole interactions between the peptide analytes and electrolyte ions. The amino acid sequences of the individual components were derived from MS(n) using the doubly or triply charged pseudomolecular ions as well as characteristic fragments as precursor ions. The exchange of Ala by alpha-aminoisobutyric acid (Aib) which is frequently observed for peptaibols was detected for several components. Additional variations included the exchange of Gln to Glu, and the loss of the C-terminal amino alcohol or of the first six amino acids from the N-terminus with concomitant formation of pyroglutamyl residues. In most cases comigration of the Aib peptaibols with the respective Ala component was observed as the mass difference of 14 Da as the result of the amino acid exchange was not sufficient to translate into an electrophoretic separation under the conditions applied. However, proper selection of the precursor ions allowed the unequivocal analysis of the components. Additional TOF-MS measurements were performed in order to resolve the ammonium adducts from comigrating compounds (i.e., Aib-Ala exchange) and to confirm the amino acid composition of the individual components. Except for neutral compounds migrating close to the EOF the mass accuracy was better than 4 ppm for the doubly charged pseudomolecular ions and better than 2 ppm for triply charged ions.

Alamethicin↗

PubMed1988

Microorganisms associated with mouldiness of dried yam chips and their prevention.

The broad objective of this study was to isolate and identify the microorganisms causing mouldiness of stored yam chips and to look for ways of preventing the problem. Microorganisms isolated included Aspergillus flavus, A. glaucus, A. nidulans, A. niger, A. ochraceous, A. tamarii, A. candidus, Penicillium oxalicum, Trichoderma longibrachyatum, Rhizopus nigricans, Cylindrocarpon radicicola, Neurospora crassa, Botryodiplodia theobromae, Bacillus subtilis, Bacillus cereus, Erwinia carotovora and Serratia marcescens. Some of these microorganisms are transient invaders. Of the calcium-based chemicals used to prevent mouldiness, only CaCO3 and Ca(OH)2 proved effective and also prevented infestation of the chips by storage pests throughout the period of study. Whereas the product of the untreated yam chips was preferred in terms of colour, the product of CaCO3- and Ca(OH)2-treated yam chips were preferred in terms of taste and texture. As for physiological reaction after eating the products of the treated chips, Ca(OH)2- treated chips seemed to be more preferred by the members of the tasting panel.

Calcium Chloride↗

PubMed2006

The effect of self-designed bifunctional RGD-containing fusion protein on the behavior of human keratinocytes and dermal fibroblasts.

In this study, self-designed bifunctional RGD-containing fusion protein (BFP) was grafted on the petri dish to evaluate its cytotoxicity and attachment efficiency on primary cultured keratinocytes and dermal fibroblasts. Two lengths of the GRGDS sequences were separately fused to the N-terminus and C-terminus of the Trichoderma koningii cellobiohydrolase I gene cellulose-binding domain, to serve as linking molecule between the cell and the substrate. The grafting procedure was no more labor-intensive and could be done just in aqueous condition itself. The epidermal keratinocytes and dermal fibroblasts, harvested and separated from human foreskin, were cultured in serum-free keratinocyte culture medium and DMEM, respectively. The BFP was dissolved in double-deionized water, and was prepared at different concentrations. The BFP solution was subsequently added into the petri dish for grafting. MTT assay, total DNA measurement, and lactate dehydrogenase analysis were used to evaluate the cell viability, cell proliferation, and cytotoxicity. The immunochemical stain and SEM examination were chosen to make sure that the cultured cells still kept in phenotype. The results showed that the self-designed BFP was successfully coated on the petri dish to improve the cells' adhesion. The whole coating procedure was just done in aqueous solution without any organic solvent being involved. This method was much simpler than the traditional one, and there was no possibility to damage the immobilized biomolecules. From the results of the study, BFP could enhance attachment of keratinocytes and dermal fibroblasts without losing normal cell morphology and keep keratinocytes on the desired differentiation pathway. We believe that coating BFP on petri dish not only enhanced the keratinocyte attachment but also promoted keratinocytes proliferation. We suggest that the self-designed BFP has a great potential to apply on surface modification for the tissue-engineering scaffolds in the future.

Amino Acid Sequence↗